EP2978770B1 - Rückfaltung von proteinen - Google Patents

Rückfaltung von proteinen Download PDF

Info

Publication number
EP2978770B1
EP2978770B1 EP14775414.7A EP14775414A EP2978770B1 EP 2978770 B1 EP2978770 B1 EP 2978770B1 EP 14775414 A EP14775414 A EP 14775414A EP 2978770 B1 EP2978770 B1 EP 2978770B1
Authority
EP
European Patent Office
Prior art keywords
gcsf
refolding
inclusion bodies
solubilization
oxidized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP14775414.7A
Other languages
English (en)
French (fr)
Other versions
EP2978770A2 (de
EP2978770A4 (de
Inventor
Bharata Ratnam PARAYITAM
Ravikant DEVAKATE
Neeraj NARAYANAN
Gopinath GOVINDARAJAN
Vivek ARTHANARI
Jaby Jacob
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dr Reddys Laboratories Ltd
Original Assignee
Dr Reddys Laboratories Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr Reddys Laboratories Ltd filed Critical Dr Reddys Laboratories Ltd
Publication of EP2978770A2 publication Critical patent/EP2978770A2/de
Publication of EP2978770A4 publication Critical patent/EP2978770A4/de
Application granted granted Critical
Publication of EP2978770B1 publication Critical patent/EP2978770B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF

Definitions

  • the present invention relates to a refolding process for Granulocyte Colony Stimulating Factor (GCSF) from inclusion bodies.
  • GCSF Granulocyte Colony Stimulating Factor
  • CSFs colony stimulating factors
  • GCSF Granulocyte Colony Stimulating Factor
  • GCSF is one of several proteins produced by recombinant DNA technology for therapeutic use.
  • lenograstim the glycosylated form of GCSF
  • mammalian cells the glycosylated form of GCSF
  • filgrastim the non-glycosylated form is expressed in Escherichia coli ( E. coli ).
  • Proteins expressed by recombinant DNA methods in bacteria such as E.coli are usually expressed as insoluble aggregates called inclusion bodies. These protein aggregates are separated, solubilized in the presence of denaturing agents. Solubilized protein is then refolded in presence of oxidizing agents. The refolded protein can then be further purified by appropriate column chromatographic procedures.
  • GCSF A typical production process for GCSF is described in US5849883 .
  • Recombinant GCSF is obtained by lysing E. coli cells and separating out the inclusion bodies containing GCSF. Inclusion bodies are then solubilized with deoxycholate and the extracted GCSF is refolded in the presence of a denaturant agent and an oxidizing agent. Refolded protein is further purified using two ion exchange chromatography steps.
  • EP0547102 explains a process of solubilization of proteins by addition of cationic surfactant and providing conditions to allow for refolding.
  • EP1630173 teaches a method for solubilization of GCSF from inclusion bodies by using reduced glutathione and a denaturing agent for solubilizing inclusion bodies.
  • US7538198 and EP1434789 provide a process of refolding of a protein by subjecting a protein aggregate and a denaturing agent to increased pressure and removing the dissolved protein from the increased pressure to fold the protein.
  • US2011294990 explains a process of refolding a protein by using two chaotropic agents and US2011034678 describes a process of refolding of a protein by using guanidine in the refolding buffer.
  • WO2011113601 teaches a process for refolding of GCSF by using oxidized glutathione and at least one reversed phase chromatography.
  • GCSF obtained by solubilization and refolding of inclusion bodies, contains the native, oxidized and the reduced forms of GCSF.
  • the reduced forms of GCSF are reported to form aggregates, whereas the oxidized form of GCSF have reduced bioactivity ( Reubsaet et al., J. Pharm. Biomed. Anal. 17, 283-289, 1998 ).
  • a chromatographic step is used downstream of refolding which caters to removal of the oxidized impurities generated during solubilization and refolding.
  • the current invention provides a method to reduce the generation of oxidized impurities by an optimized solubilization process during refolding of GCSF.
  • the present invention discloses a method for refolding of recombinant GCSF that minimizes the generation of oxidized forms of GCSF by optimizing the solubilization of inclusion bodies containing recombinant GCSF.
  • the invention discloses a method for optimal time for solubilization of inclusion bodies during the refolding process to improve the yield of correctly folded form of GCSF with significant reduction in oxidized forms generated during the process.
  • FiG 1 Effect of incubation time, prior to adding refolding buffer, on solubilized inclusion bodies (at pH 12). Oxidized forms of GCSF (out-put parameter), is measured in refolded protein solution.
  • inclusion bodies Proteins expressed by recombinant DNA methods in prokaryotic systems such as E. coli, are usually expressed as insoluble aggregates called inclusion bodies which require denaturation and renaturation (refolding) in order to recover the correctly folded biologically active form.
  • inclusion bodies refer to the insoluble aggregates of proteins expressed by recombinant DNA methods in microbial expression systems.
  • refolding buffer refers to a buffer that is used in renaturation or refolding of the protein of interest.
  • the present invention provides a method for refolding GCSF obtained from inclusion bodies, wherein oxidized form of GCSF is reduced to less than 1.5%.
  • the invention provides a method of refolding GCSF obtained from inclusion bodies by solubilization at about pH 11 to 12 wherein there is no incubation after the solubilization step and wherein the oxidized form of GCSF is reduced to less than 1.5%.
  • the invention provides a method for reducing the oxidized form of Granulocyte Colony Stimulating Factor (GCSF) obtained from inclusion bodies, comprising the steps of;
  • GCSF Granulocyte Colony Stimulating Factor
  • the invention provides a method of refolding GCSF obtained from inclusion bodies by solubilization at about pH 11 to 12 wherein there is no incubation after the solubilization step and prior to adding the refolding buffer wherein the oxidized form of GCSF is reduced to less than 1.5%.
  • Cells containing recombinant GCSF in the form of inclusion bodies are resuspended in phosphate buffered saline (PBS buffer) in the ratio of 5 mL PBS buffer per gram of cell pellet.
  • PBS buffer phosphate buffered saline
  • the cell suspension in PBS buffer is stirred on a magnetic stirrer for 20 min to make a homogenous solution.
  • the cell suspension is centrifuged at a relative centrifugal force (RCF) of 13000 for 30 min at a temperature of 4 °C. After centrifugation, supernatant is discarded and the pellet is resuspended in lysis buffer (50 mM Tris and 10 mM EDTA) in the ratio of 10 ml lysis buffer per gram of pellet.
  • the cell suspension in lysis buffer is stirred gently on a magnetic stirrer for 20 min.
  • the cell suspension is passed through the homogenizer two times at a pressure of 900-1000 bar till a drop in OD 600 equivalent to 70% is achieved.
  • the cell lysate is collected and centrifuged at 13000 RCF for 30 min at 4 °C.
  • the pellet obtained is of the inclusion bodies.
  • Inclusion bodies obtained from example 1 are solubilized with 8 M urea and water for injection (WFI). The pH of this suspension is adjusted to 11 to 13 by adding small quantities of 1 N sodium hydroxide solution.
  • solubilization mixture from example 2 is diluted 20 times by directly adding the refolding buffer (25 mM Tris, 1 mM EDTA and 0.6 M Arginine and 5% Sorbitol). The pH is adjusted to 8.6 - 9.4 by Glacial Acetic Acid at pH 9.0.
  • the refolding mixture from example 3 is subject to redox shuffling by addition of 0.2 mM cysteine, after a 10 minute interval 1.8 mM cystine is added which is again followed by a 10 minute interval after which 0.2 mM cysteine is added. This mixture is incubated at 2-8°C for 16 hours to obtain refolded GCSF.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Claims (1)

  1. Verfahren zum Reduzieren der oxidierten Form des von Einschlusskörpern erhaltenen Granulozyten-Koloniestimulierenden Faktors (GCSF), das die folgenden Schritte beinhaltet:
    a) Solubilisieren von Einschlusskörpern in einem Denaturierungsmittel, das 8 M Harnstoff, pH 11 bis pH 13, umfasst, wobei auf den Solubilisierungsschritt unmittelbar (ohne Inkubation) Schritt (b) folgt,
    b) Zugeben eines Rückfaltungspuffers, der eine basische Aminosäure und einen Polyalkohol umfasst, pH von 8,6 bis 9,4, zum solubilisierten Gemisch von Schritt (a),
    c) Zugeben eines Redox-Shuffling-Gemischs, das 0,2 mM Cystein und 1,8 mM Cystin umfasst, zum Rückfaltungsgemisch von Schritt (b) und Inkubieren für 16 Stunden,
    wobei das genannte Verfahren die oxidierten Formen von GCSF auf weniger als 1,5 % reduziert.
EP14775414.7A 2013-03-29 2014-03-28 Rückfaltung von proteinen Active EP2978770B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1431CH2013 2013-03-29
PCT/IB2014/060251 WO2014155349A2 (en) 2013-03-29 2014-03-28 Refolding of proteins

Publications (3)

Publication Number Publication Date
EP2978770A2 EP2978770A2 (de) 2016-02-03
EP2978770A4 EP2978770A4 (de) 2016-10-26
EP2978770B1 true EP2978770B1 (de) 2019-11-06

Family

ID=51625560

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14775414.7A Active EP2978770B1 (de) 2013-03-29 2014-03-28 Rückfaltung von proteinen

Country Status (3)

Country Link
US (1) US9982012B2 (de)
EP (1) EP2978770B1 (de)
WO (1) WO2014155349A2 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016116947A1 (en) * 2015-01-21 2016-07-28 Indian Institute Of Technology A coiled flow inverter reactor for continuous refolding of denatured recombinant proteins and other mixing operations

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE68929566D1 (de) 1988-05-13 2010-01-28 Amgen Inc Verfahren zur Isolierung und Reinigung von G-CSF
CA2090701C (en) 1990-09-05 2000-05-16 Southern Cross Biotech Pty. Ltd. Solubilization of proteins in active forms
US6583268B2 (en) * 2000-01-25 2003-06-24 Oklahoma Medical Research Foundation Universal procedure for refolding recombinant proteins
WO2002062827A2 (en) 2000-10-31 2002-08-15 The Regents Of The University Of Colorado, A Body Corporate Improved protein disaggregation and refolding using high pressure
WO2003102013A2 (en) * 2001-02-23 2003-12-11 Gonzalez-Villasenor Lucia Iren Methods and compositions for production of recombinant peptides
EP1449848A1 (de) * 2003-02-20 2004-08-25 GBF German Research Centre for Biotechnology Verfahren zur Herstellung von Proteinen aus der Cystine-Knoten-Familie
DE102004041639A1 (de) 2004-08-27 2006-03-02 Bioceuticals Arzneimittel Ag Verfahren zur Gewinnung von biologisch aktivem humanen G-CSF aus Inclusion Bodies
RU2439076C2 (ru) 2006-07-14 2012-01-10 Дженентек, Инк. Способ выделения повторно свернутого рекомбинантного белка из культуры прокариотических клеток (варианты)
WO2008096370A2 (en) * 2007-02-05 2008-08-14 Natco Pharma Limited An efficient and novel purification method of recombinant hg-csf
WO2010105227A1 (en) 2009-03-13 2010-09-16 Aerovance, Inc. Methods of renaturation of recombinant proteins
US20120093765A1 (en) 2009-06-16 2012-04-19 Lupin Limited Process for purification of recombinant human granulocyte colony stimulating factor
WO2011113601A1 (en) 2010-03-17 2011-09-22 Biogenerix Ag Method for obtaining biologically active recombinant human g-csf
WO2014128726A2 (en) * 2013-02-22 2014-08-28 Biogenomics Limited Process for high efficiency refolding of recombinant proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
US9982012B2 (en) 2018-05-29
US20160031932A1 (en) 2016-02-04
WO2014155349A3 (en) 2015-04-09
EP2978770A2 (de) 2016-02-03
WO2014155349A2 (en) 2014-10-02
EP2978770A4 (de) 2016-10-26

Similar Documents

Publication Publication Date Title
US9458207B2 (en) Methods for refolding G-CSF from inclusion bodies
JPS611393A (ja) Ｉｌ−２の回収及び精製方法
WO2008096370A2 (en) An efficient and novel purification method of recombinant hg-csf
JPS6272625A (ja) 安定な経口投与剤及びその製造方法
JP2011078421A (ja) 封入体における高い量の正しく折りたたまれた異種タンパク質の生産方法
US10519209B2 (en) Process for purification of rHu-GCSF
US20120093765A1 (en) Process for purification of recombinant human granulocyte colony stimulating factor
US20160060291A1 (en) Process for renaturation of polypeptides
EP2978770B1 (de) Rückfaltung von proteinen
US9416164B2 (en) Method for the production of polypeptides
EP2341061B1 (de) Neuartiges Verfahren zur Herstellung von G-CSF
US20150376228A1 (en) Process for high efficiency refolding of recombinant proteins
WO2017177981A1 (zh) 一种重组人粒细胞刺激因子的复性及纯化方法
US20180037604A1 (en) Process for the purification of recombinant proteins
EP2532734B1 (de) Verfahren für die herstellung von interferon alpha 5
WO2020234742A1 (en) Granulocyte colony stimulating factor purification
WO2019077432A1 (en) IMPROVED PURIFICATION METHOD OF RECOMBINANT PTH (1-34)
CN104120159A (zh) 一种重组人粒细胞集落刺激因子的制备方法
TW202106698A (zh) 用於製備粒細胞群落刺激因子的方法
EP1833846A2 (de) Verfahren zur herstellung eines therapeutisch reinen granulozytenmacrophagen-koloniestimulierenden faktors
EA023379B1 (ru) ШТАММ ESCHERICHIA COLI BL21(DE3)-pIE-GAMMA И СПОСОБ ПОЛУЧЕНИЯ ПРЕПАРАТА НАТИВНОГО РЕКОМБИНАНТНОГО ИНТЕРФЕРОНА ГАММА ЧЕЛОВЕКА

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20150925

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20160927

RIC1 Information provided on ipc code assigned before grant

Ipc: C07K 14/535 20060101ALI20160921BHEP

Ipc: C07K 1/113 20060101ALI20160921BHEP

Ipc: C12N 15/09 20060101ALI20160921BHEP

Ipc: C07K 1/36 20060101AFI20160921BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20170905

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20190607

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

Ref country code: AT

Ref legal event code: REF

Ref document number: 1198602

Country of ref document: AT

Kind code of ref document: T

Effective date: 20191115

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602014056343

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20191106

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200206

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200207

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200206

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200306

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200306

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602014056343

Country of ref document: DE

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1198602

Country of ref document: AT

Kind code of ref document: T

Effective date: 20191106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20200807

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20200331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200328

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200328

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20210324

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20210209

Year of fee payment: 8

Ref country code: DE

Payment date: 20210310

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191106

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602014056343

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20220328

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220328

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220331

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221001